Bioneer Corporation 8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea Tel: Fax:

Transcription

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2 Bioneer Corporation 8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea Tel: Fax:

3 Contents I. Overview II. Kit components III. Storage IV. Intended use V. Safety warnings and precautions VI. Warranty and liability VII. Technical assistance VIII. Quality management IX. Product specifications Small Amount of Sample X. Principle XI. Materials and Equipment Needed But Not Provided XII. Protocols Before You Begin 6 DNA Extraction from Whole Blood 6 DNA Extraction from Cultured Cell 10 DNA Extraction from Animal Tissue 14 DNA Extraction from Gram-Negative Bacteria 18 DNA Extraction from Gram-Positive Bacteria 19 DNA Clean-Up 20 XIII. Troubleshooting guide XIV. Ordering information XV. Explanation symbols

4 I. Overview Description MagListo TM 5M Genomic DNA Extraction Kit utilizes the magnetic bead approach to extract total DNA from a variety of sources, such as whole blood, animal tissue and cultured cell using Magnetic Nano Beads and MagListo TM Magnetic Separation Rack. The use of MagListo TM Magnetic Separation Rack along with the kit greatly increases user convenience by saving process time without the need of centrifuge. Features and Benefits - Magnetic Nano Beads enable rapid extraction. - No costly capital instrument to instruments except MagListo TM Magnetic Separation Rack. Applications Gene Cloning, PCR, Real-Time PCR, Southern Blotting, SNP genotyping II. Kit Components MagListo TM 5M Genomic DNA Extraction Kit Cat. no. 3602, 3603 *K-3602 (8 reaction) **K-3603 (100 reaction) Buffer 1 (Lysis) 2 ml x 1 ea 25 ml x 1 ea Buffer 2 (Binding) 2 ml x 1 ea 25 ml x 1 ea Buffer 3 (1 st Washing) 6 ml x 1 ea 60 ml x 1 ea Buffer 4 (2 nd Washing) 1.6 ml x 1 ea 16 ml x 1 ea Buffer 5 (Elution) 1 ml x 1 ea 25 ml x 1 ea Magnetic Nano Bead - DNA 1 ml x 1 ea 1.8 ml x 6 ea Proteinase K powder, lyophilized 5 mg x 1 ea 25 mg x 2 ea RNase A powder, lyophilized 6 mg x 1 ea 24 mg x 2 ea *Mini - 8rxn, Midi - 1rxn **Mini 100 rxn, Midi 15 rxn, Maxi 8 rxn 1

5 III. Storage MagListo TM 5M Genomic DNA Extraction Kit should be stored dry at room temperature. It can be stored for up to 2 years if it remains sealed. MagListo TM 5M Genomic DNA Extraction Kit provides optimized Buffer 2 (Binding) and Buffer 3 (1 st Washing) which is poisonous and hazardous. Please, wear gloves and goggle eye protection when working with Buffer 2 (Binding) and Buffer 3 (1 st Washing). IV. Intended Use MagListo 5M Genomic DNA Extraction Kit is intended for research use only. This kit is not intended for human or veterinary diagnostics. V. Safety Warnings and Precautions Please inquire BIONEER s Customer Service Center to obtain a copy of the Material Safety Data Sheet (MSDS) for this product. Before, during and after use of this kit as described in this User s Guide, all potentially hazardous materials (i.e. materials that may have come in contact with genetically recombinant samples) including tubes, tips and materials should be processed and disposed of according to applicable and appropriate regulations of the municipality/government in which this product is being used. A user must also be equipped with basic experimental techniques required for correct execution of the experiments described in this User s Guide. Some applications that may be performed with this kit may infringe upon existing patents in certain countries. The purchase of this kit does not include or provide a license to perform patented applications. Users may be required to obtain a license depending on country and application. We do not condone nor recommend the unlicensed use of a patented application. VI. Warranty and Liability All BIONEER products are manufactured and tested under strict quality control protocols. BIONEER guarantees the quality of all directly manufactured products during the warranty period of one (1) year from the date of purchase. If any issues are discovered relating to compromise in product quality, immediately contact BIONEER s Customer Service Center (sales@bioneer.com). 2

6 BIONEER does not assume liability for misuse of the product, i.e. usage of the product for any purposes other than its intended purpose as described in the User s Guide. BIONEER assumes liability under the condition that users disclose all information related to the problem to BIONEER in written form within 30 days of occurrence. VII. Technical Assistance At Bioneer, we pride ourselves on being responsive to your needs. If you have any questions or would like to find out more information about MagListo products, please contact us. We look forward to hearing from you! Technical Support For all technical questions and troubleshooting on Bioneer products and applications. Tel: sales@bioneer.co.kr In North America Tel: support@bioneer.us.com VIII. Quality Management Every aspect of our quality management system from product development, production to quality assurance and supplier qualification meets the world-class standards. Each lot of MagListo 5M Genomic DNA Extraction Kit is carefully tested by the quality control team. 3

7 IX. Product Specifications Sample Type Mini (Typical yield) Midi (Typical yield) Maxi (Typical yield) Whole blood 200 μl ( < 10 μg) 2 ml ( < 80 μg) 4 ml ( < 150 μg) Cultured cell ~ 1 x 10 6 ( < 12 μg) ~ 5 x 10 6 (< 60 μg) ~ 1 x 10 7 (< 120 μg) Animal tissue ~ 25 mg ( < 10 μg) ~ 100 mg ( < 40 μg) ~ 250 mg (< 120 μg) Bacteria (Gram (-), (+)) ~ 1 x 10 9 (< 15 μg) ~ 5 x 10 9 (< 80 μg) ~ 1 x (< 150 μg) Expected purity A 260 / 280 > 1.8, A 260 / 230 > 1.4 * The yield of low copy cell could be less than the figures shown in the table. ** For cultured cells, lower cell numbers can be used to a minimal cell number of ~10 4 for micro scale extraction. Small amount of sample MagListo TM 5M Genomic DNA Extraction Kit is also able to extract genomic DNA from a small quantity of sample. If the sample is low copy cell (< 1x10 4 ) or has a small amount of DNA, we recommend that about 4 μg of carrier DNA (a homopolymer such as poly-da, poly-dt, or gdna) or RNA should be added to the starting material. Ensure that the carrier DNA does not interfere with your downstream application. Carrier RNA can be removed later by RNase digestion. Refer to DNA Extraction from Cultured Cell for Micro which is optimized protocol for extraction of DNA from low copy cell in page 10 X. Principle MagListo TM 5M Genomic DNA Extraction Kit is designed for the extraction of genomic DNA from a variety of sources including high molecular weight up to 40 Kb (Note: this is often for most DNA based application). The overall principle is based on adsorption of DNA onto the magnetic nano bead by chaotropic salt. For instance, chaotropic agents in Buffer 2 (Binding) contains guanidine hydrochloride, as which removes water molecules around DNA and silica coated magnetic bead surface resulting in genomic DNA then being captured by magnetic beads. The magnetic nano bead and nucleic acid complexes are pulled and fixed on the tube wall using a magnetic force, followed by washing with ethanol 4

8 to remove debris and excessive salts. The captured nucleic acids are then eluted by Buffer 5 (Elution), an aqueous solution with optimal ph. Sample Lysis Binding Washing Elution XI. Materials and Equipment Needed But Not Provided ml or 2 ml tube, 15 ml tube, 50 ml tube 2. Vortex mixer 3. Absolute ethanol 4. Thermal block or dry oven 5. Phosphate buffered saline (PBS) 6. Blow dryer or heat gun 7. MagListo TM Magnetic Separation Rack Magnetic Separation Rack Choice Tube 1 ml tube with 8-cap strip 1.5 ml or 2 ml microcentrifuge tube 15 ml tube 50 ml tube MagListo TM Magnetic Separation Rack MagListo TM -8Ch Magnetic Separation Rack (Cat. no. TM-1000) MagListo TM -2 Magnetic Separation Rack (Cat. no. TM-1010) MagListo TM -15 Magnetic Separation Rack (Cat. no. TM-1020) MagListo TM -50 Magnetic Separation Rack (Cat. no. TM-1030) (Note) Please refer to the ordering information table on the latter part of the manual which contains the appropriate catalog number for specific size of tubes. 5

9 XII. Protocols Before you begin 1. Completely dissolve Proteinase K (KB-0111) in 1,250 ul of nuclease-free water. Dissolved Proteinase K should be stored at Completely dissolve RNase A (KB-3101) in 600 ul of nuclease-free water. Dissolved RNase A should be stored at Buffer 2 (Binding) contains chaotropic salt. You should take the appropriate laboratory safety precautions and wear gloves when handling. 4. Add the correct amount of absolute ethanol to Buffer 3 (1 st Washing) and Buffer 4 (2 nd Washing). a. DNA Extraction from Whole Blood for Mini/Midi/Maxi scale 1. Add 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of proteinase K (see Before you begin ) to each specific tube format. a. (Mini) Add proteinase K to a 1.5 ml or 2 ml tube. b. (Midi) Add proteinase K to a 15 ml tube. c. (Maxi) Add proteinase K to a 50 ml tube. 2. Apply 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) of whole blood and buffy coat to the tube containing proteinase K. (Note) If the sample volume is less than indicated volume above, make the total volume 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) by adding 1X PBS to achieve maximum lysis efficiency and yield. 3. (Lysis: 3-4) Add 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) of Buffer 2 (Binding) to each sample and mix immediately and thoroughly using a vortex mixer. You must completely resuspend the sample to achieve maximum lysis efficiency. 4. Incubate at 60 for 10 min. 5. (DNA Precipitation) Add 400 μl (mini)/ 4 ml (midi)/ 8 ml (maxi) of absolute ethanol and mix well using a 6

10 vortex mixer or pipetting. 6. (DNA binding with Magnetic Nano Bead: 6-8) Add 100 μl (mini)/ 500 μl (midi)/ 1 ml (maxi) of Magnetic Nano Bead solution to the tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. (Note) Magnetic Nano Bead solution contains magnetic nano beads. Please shake well before use. 7. Place the tube in MagListo TM -2 (mini)/ MagListo TM -15 (midi)/ MagListo TM -50 (maxi) Magnetic Separation Rack with the magnet plate attached and invert the tube 3~4 times gently until the beads tightly bind to the magnet. - Attachment Combine the magnet plate to the stand. 8. Without removing the tube from MagListo TM, carefully pour the supernatant out and completely remove the remaining supernatant using a paper towel. In process, the magnetic crude pellet remains attached to the side of tube. - Discard solution Discard solution by inverting the MagListo rack. The silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discard solution, invert the rack completely for the solution not to smear on the rack. 9. (1 st washing: 9-12) Detach the magnet plate from MagListo TM rack. Add 500 μl (mini)/ 3 ml (midi)/ 5 ml (maxi) of Buffer 3 (1 st Washing) to the tube. Close the cap and mix by vortex mixer until the beads are fully resuspended. 7

11 - Detachment Push up the magnet plate gently. 10. Attach the magnet plate to MagListo TM rack and invert the tube 3 ~ 4 times gently until the beads tightly bind to the magnet. 11. Without removing the tube from MagListo TM rack, pour the supernatant out and remove the remaining supernatant using a paper towel by blotting. 12. Repeat the above step 9 ~ 11 by adding 500 μl (mini)/ 3 ml (midi)/ 6 ml (maxi) of Buffer 3 (1 st Washing) for additional washing to remove pigment and other debris. 13. (2 nd washing) Repeat the above step 9 ~ 11 by adding 700 μl (mini)/ 5 ml (midi)/ 10 ml (maxi) of Buffer 4 (2 nd Washing) for additional washing. 14. (3 rd washing) Repeat the above step 9 ~ 11 by adding 700 μl (mini)/ 5 ml (midi)/ 10 ml (maxi) of absolute ethanol for additional washing. 15. (Drying) Completely dry the beads with the tube open and use a heat gun or a blow dryer for 1 min (mini)/ 3 min (midi)/ 5 min (maxi) 3 cm away from the top of the tube. (Note) Without using a heat gun or a blow dryer, place the rack lying down in a dry oven at 60 for 10 min (mini)/ 20 min (midi)/ 30 min (maxi). 16. (Elution: 16-20) Add 100 μl (mini)/ 500 μl (midi)/ 1 ml (maxi) of Buffer 5 (Elution) or distilled water to the tube with the magnet plate detached and resuspend completely by pipetting or vortex mixer for 15 sec. 17. Incubate the tube at 60 for 1 min. 8

12 18. Attach the magnet plate to MagListo TM rack and invert the tube 3 ~ 4 times gently until the beads tightly bind to the magnet. 19. Without removing the tube from MagListo TM rack, carefully take the supernatant containing DNA to a sterile microcentrifuge tube. 20. Discard the used magnetic nano beads. Do not reuse the beads. Summary of Reagents Volume in Each Step of DNA Extraction from Whole Blood Step Buffer Mini Midi Maxi Page Blood (+ PBS) 200 μl 2 ml 4 ml P. 6 Lysis Buffer 2 (Binding) 200 μl 2 ml 4 ml P. 6 Precipitation Absolute Ethanol 400 μl 4 ml 8 ml P. 6 Bead Binding Magnetic Nano Bead - DNA 100 μl 500 μl 1 ml P. 6 1 st Washing (Repeat) Buffer 3 (1 st Washing) 500 μl 3 ml 6 ml P. 7 2 nd Washing Buffer 4 (2 nd Washing) 700 μl 5 ml 10 ml P. 8 3 rd Washing Absolute Ethanol 700 μl 5 ml 10 ml P. 8 Elution Buffer 5 (Elution) 100 μl 500 μl 1 ml P. 8 9

13 b. DNA Extraction from Cultured Cell for Micro/Mini/Midi/Maxi 1. Centrifuge the cultured cells (~ 1x10 4 (micro)/ ~ 1x10 6 (mini)/ ~ 5x10 6 (midi)/ ~ 1x10 7 (maxi)) for 10 min at 300 x g. Discard supernatant carefully. (Note) For cultured cells, micro scale is included to accommodate the use of lower cell number than 1x10 4 cells. Thus, given volume amount of this kit component is adjusted and mentioned on this procedure. 2. Resuspend the pellet in 100 μl (micro)/ 200 μl (mini)/ 1 ml (midi, maxi) of 1X PBS and transfer them to each specific tube format. a. (Micro/Mini) Transfer the resuspended pellet to a 1.5 ml or 2 ml tube. b. (Midi/Maxi) Transfer the resuspended pellet to a 15 ml tube. 3. Add 10 μl (micro)/ 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of proteinase K (see Before you begin ) to the tube. 4. If RNA-free genomic DNA is required, add up to 2 μl (micro)/ 10 μl (mini)/ 75 μl (midi), 150 μl (maxi) of RNase A (see Before you begin ) and incubate for 5 min at room temperature. 5. (Lysis: 5-6) Add 100 μl (micro)/ 200 μl (mini)/ 1 ml (midi, maxi) of Buffer 2 (Binding) to the sample and mix immediately and thoroughly using a vortex mixer. You must completely resuspend the sample to achieve maximum lysis efficiency. 6. Incubate at 60 for 10min. 7. (DNA precipitation) Add 200 μl (micro)/ 400 μl (mini)/ 2 ml (midi, maxi) of absolute ethanol and mix well by vortex mixer or pipetting. 8. (DNA binding with Magnetic Nano Bead: 8-10) Add 100 μl (micro, mini)/ 500 μl (midi)/ 1 ml (maxi) of Magnetic Nano Bead solution to the tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. (Note) Magnetic Nano Bead Solution contains magnetic nano beads. Please shake well before use. 10

14 9. Place the tube in MagListo TM -2 (micro, mini)/ MagListo TM -15 (midi, maxi) Magnetic Separation Rack with the magnet plate attached and invert the tube 3~4 times gently until the beads tightly bind to the magnet. - Attachment Combine the magnet plate to the stand. 10. Without removing the tube from MagListo TM rack, carefully pour the supernatant out and completely remove the remaining supernatant using a paper towel by blotting. - Discard solution Discard solution by inverting the MagListo rack. The silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discard solution, invert the rack completely for the solution not to smear on the rack. 11. (1 st washing: 11-13) Detach the magnet plate from MagListo TM rack. Add 700 μl (micro, mini)/ 5 ml (midi)/ 10 ml (maxi) of Buffer 3 (1 st Washing) to the tube. Close the cap and mix by vortex mixer until the beads are fully resuspended. - Detachment Push up the magnet plate gently. 12. Attach the magnet plate to MagListo TM stand and invert the tube 3~4 times gently until the beads 11

15 tightly bind to the magnet. 13. Without removing the tube from MagListo TM rack, pour the supernatant out and remove the remaining supernatant using a paper towel by blotting. 14. (2 nd washing) Repeat the above step 11 ~ 13 by adding 700 μl (micro, mini)/ 5 ml (midi)/ 10 ml (maxi) Buffer 4 (2 nd Washing) for additional washing. 15. (3 rd washing) Repeat the above step 11 ~ 13 by adding 700 μl (micro, mini)/ 5 ml (midi)/ 10 ml (maxi) absolute ethanol for additional washing. 16. (Drying) Completely dry the beads with the tube open and use a heat gun or a blow dryer for 1 min (micro, mini)/ 3 min (midi)/ 5 min (maxi) 3 cm away from the top of the tube. (Note) Without using a heat gun or a blow dryer, place the rack lying down in a dry oven at 60 for 10 min (micro, mini)/ 20 min (midi)/ 30 min (maxi). 17. (Elution: 17-21) Add 50 μl (micro)/ 100 μl (mini)/ 500 μl (midi)/ 1 ml (maxi) of Buffer 5 (Elution) or distilled water to the tube with the magnet plate detached and resuspend completely by pipetting or vortex mixer for 15 sec. 18. Incubate the tube at 60 for 1 min. 19. Attach the magnet plate to MagListo TM rack and invert the tube 3~4 times gently until the beads tightly bind to the magnet. 20. Without removing the tube from MagListo TM rack, carefully take the supernatant containing DNA to a sterile microcentrifuge tube. 21. Discard the used magnetic nano beads. Do not reuse the beads. 12

16 Summary of Reagents Volume in Each Step of DNA Extraction from Cultured Cell Step Buffer Micro Mini Midi Maxi Page Cultured Cell ~ 1 x 10 4 ~ 1 x 10 6 ~ 5 x 10 6 ~1 x 10 7 P. 10 Lysis Buffer 2 (Binding) 100 μl 200 μl 1 ml 1 ml P. 10 DNA Precipitation Absolute Ethanol 200 μl 400 μl 2 ml 2 ml P. 10 Bead Binding Magnetic Nano Bead - DNA 100 μl 100 μl 500 μl 1 ml P st Washing Buffer 3 (1 st Washing) 700 μl 700 μl 5 ml 10 ml P nd Washing Buffer 4 (2 nd Washing) 700 μl 700 μl 5 ml 10 ml P rd Washing Absolute Ethanol 700 μl 700 μl 5 ml 10 ml P. 12 Elution Buffer 5 (Elution) 50 μl 100 μl 500 μl 1 ml P

17 c. DNA Extraction from Animal Tissue for Mini/Midi/Maxi 1. (Homogenization) Disrupt (or homogenize) the sample (~ 25 mg (mini)/ ~ 100 mg (midi)/ ~ 250 mg (maxi)) with a mortar and pestle. Place them to each specific tube format. a. (Mini) Place the homogenized tissue to a 1.5 ml or 2 ml tube. b. (Midi) Place the homogenized tissue to a 15 ml tube. c. (Maxi) Place the homogenized tissue to a 50 ml tube. (Note) Immediately place the weighted, fresh or frozen tissue in liquid nitrogen and grind to a fine powder with mortar and pestle under liquid nitrogen. Final yield of DNA depends on the amount and the type of the used tissue. 2. (Lysis: 2-6) Add 180 μl (mini)/ 1.8 ml (midi)/ 3.6 ml (maxi) of Buffer l (Lysis). 3. Add 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of proteinase K (see Before you begin ) to the tube and mix thoroughly using a vortex mixer. 4. If RNA-free genomic DNA is required, add up to 10 μl (mini)/ 75 μl (midi)/ 150 μl (maxi) of RNase A (see Before you begin ) and incubate for 2 min at room temperature. 5. Incubate at 60 until the tissue is completely lysed. 6. Add 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) of Buffer 2 (Binding) to the tube and mix immediately and thoroughly using a vortex mixer. You must completely resuspend the sample to achieve maximum lysis efficiency. 7. (DNA precipitation) Add 400 μl (mini)/ 4 ml (midi)/ 8 ml (maxi) of absolute ethanol and mix well using a vortex mixer or pipetting. 8. (DNA binding with Magnetic Nano Bead: 8-10) Add 100 μl (mini)/ 500 μl (midi)/ 1 ml (maxi) of Magnetic Nano Bead solution to the tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. (Note) Magnetic Nano Bead solution contains magnetic nano beads. Please shake well before use. 14

18 9. Place the tube in MagListo TM -2 (mini)/ MagListo TM -15 (midi)/ MagListo TM -50 (maxi) Magnetic Separation Rack with the magnet plate attached and invert the tube 3~4 times gently until the beads tightly bind to the magnet. - Attachment Combine the magnet plate to the stand. 10. Without removing the tube from MagListo TM rack, carefully pour the supernatant out and completely remove the remaining supernatant using a paper towel by blotting. - Discard solution Discard solution by inverting the MagListo rack. The silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discard solution, invert the rack completely for the solution not to smear on the rack. 11. (1 st washing: 11-13) Detach the magnet plate from MagListo TM rack. Add 700 μl (mini)/ 5 ml (midi)/ 10 ml (maxi) of Buffer 3 (1 st Washing) to the tube. Close the cap and mix by vortex mixer until the beads are fully resuspended. - Detachment Push up the magnet plate gently. 15

19 12. Attach the magnet plate to MagListo TM stand and invert the tube 3 ~ 4 times gently until the beads tightly bind to the magnet. 13. Without removing the tube from MagListo TM rack, pour the supernatant out and remove the remaining supernatant using a paper towel by blotting. 14. (2 nd washing) Repeat the above step 11 ~ 13 by adding 700 μl (mini)/ 5 ml (midi)/ 10 ml (maxi) Buffer 4 (2 nd Washing) for additional washing. 15. (3 rd washing) Repeat the above step 11 ~ 13 by adding 700 μl (mini)/ 5 ml (midi)/ 10 ml (maxi) absolute ethanol for additional washing. 16. (Drying) Completely dry the beads with the tube open and use a heat gun or a blow dryer for 1 min (mini)/ 3 min (midi)/ 5 min (maxi) 3 cm away from the top of the tube. (Note) Without using a heat gun or a blow dryer, place the rack lying down in a dry oven at 60 for 10 min (mini)/ 20 min (midi)/ 30 min (maxi). 17. (Elution: 17-21) Add 100 μl (mini)/ 500 μl (midi)/ 1 ml (maxi) of Buffer 5 (Elution) or distilled water to the tube with the magnet plate detached and resuspend by pipetting or vortex mixer for 15 sec. 18. Incubate the tube at 60 for 1 min. 19. Attach the magnet plate to MagListo TM rack and invert the tube 3~4 times gently until the beads tightly bind to the magnet. 20. Without removing the tube from MagListo TM rack, carefully take the supernatant containing DNA to a sterile microcentrifuge tube. 21. Discard the used magnetic nano beads. Do not reuse the beads. 16

20 Summary of Reagents Volume in Each Step of DNA Extraction from Animal Tissue Step Buffer Mini Midi Maxi Page Animal tissue ~ 25 mg ~ 100 mg ~ 250 mg P. 14 Lysis Buffer l (Lysis) 180 μl 1.8 ml 3.6 ml P. 14 Buffer 2 (Binding) 200 μl 2 ml 4 ml P. 14 DNA Precipitation Absolute Ethanol 400 μl 4 ml 8 ml P. 14 Bead Binding Magnetic Nano Bead DNA 100 μl 500 μl 1 ml P st Washing Buffer 3 (1 st Washing) 700 μl 5 ml 10 ml P nd Washing Buffer 4 (2 nd Washing) 700 μl 5 ml 10 ml P rd Washing Absolute Ethanol 700 μl 5 ml 10 ml P. 16 Elution Buffer 5 (Elution) 100 μl 500 μl 1 ml P

21 d. DNA Extraction from Gram-Negative Bacteria for Mini/Midi/Maxi 1. (Cell collection) Collect the bacteria cells (~1x10 9 (mini)/ ~5x10 9 (midi)/ ~1x10 10 (maxi)) by centrifugation at 6000 x g (> 8,000 rpm in a microcentrifuge) for 10 min (or >3,500 rpm for 20 min). And completely remove the media by pipetting. 2. (Resuspension of cell pellet: 2-3) Add 180 μl (mini)/ 1.8 ml (midi)/ 3.6 ml (maxi) of Buffer l (Lysis) to the collected cell pellet and completely resuspend by vortex mixer or pipetting. 3. Transfer the resuspended cell pellet to each specific tube format. a. (Mini) Transfer the resuspended pellet to a 1.5 ml or 2 ml tube. b. (Midi) Transfer the resuspended pellet to a 15 ml tube. c. (Maxi) Transfer the resuspended pellet to a 50 ml tube. 4. Go to step 3 of DNA Extraction from Animal Tissue in page 14 and continue the extraction process. 18

22 e. DNA Extraction from Gram-Positive Bacteria for Mini/Midi/Maxi 1. (Cell collection) Collect the bacteria cells (~ 1x10 9 (mini)/ ~ 5x10 9 (midi)/ ~ 1x10 10 (maxi)) by centrifugation at 6,000 x (>8,000 rpm in a microcentrifuge) for 10 min (or >3,500 rpm for 20 min). And completely remove the media by pipetting. 2. (Resuspension of cell pellet: 2-3) Add 180 μl (mini)/ 1.8 ml (midi)/ 3.6 ml (maxi) of Gram-Positive Lysis Buffer (not provided) to the collected cell pellet and completely resuspend by vortex mixer or pipetting. (Note) Gram-Positive Lysis Buffer can be prepared by using this formulation: 20 mm Tris-HCl (ph8.0), 2 mm sodium EDTA, 1.2% Triton X-100) 3. Transfer the resuspended cell pellet to each specific tube format. a. (Mini) Transfer the resuspended pellet to a 1.5 ml or 2 ml tube. b. (Midi) Transfer the resuspended pellet to a 15 ml tube. c. (Maxi) Transfer the resuspended pellet to a 50 ml tube. 4. (Lysis: 4-9) Add 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of lysozyme (100 mg/ml, not provided) and mix thoroughly using a vortex mixer. 5. If RNA-free genomic DNA is required, add up to 10 μl (mini)/ 75 μl (midi)/ 150 μl (maxi) of RNase A (see Before you begin ). 6. Incubate at 37 for 30 min. 7. Add 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of proteinase K (see Before you begin ). 8. Add 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) of Buffer 2 (Binding) to the tube and mix immediately and thoroughly using a vortex mixer. 9. Incubate at 60 for 30 min or until the cells are completely lysed. 10. Go to step 7 of DNA Extraction from Animal Tissue in page 14 and continue the extraction process. 19

23 f. DNA Clean-Up 1. Transfer the eluate or enzyme reaction product to each specific tube format. a. (Mini) Transfer the eluate to a 1.5 ml or 2 ml tube b. (Midi/Maxi) Transfer the eluate to a 15 ml tube 2. If RNA-free genomic DNA is required, add up to 10 μl (mini)/ 75 μl (midi)/ 150 μl (maxi) of RNase A (see Before you begin ) and incubate for 2 min at room temperature. 3. (Binding) Add 1 volume of Buffer 2 (Binding) to the eluate and mix completely by vortex mixer. 4. (DNA precipitation) Add 3 volumes of absolute ethanol to the eluate and mix well by vortex mixer. 5. (DNA binding with Magnetic Nano Bead: 5-7) Add 100 μl (mini)/ 500 μl (midi)/ 1 ml (maxi) of Magnetic Nano Bead solution to the tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. (Note) Magnetic Nano Bead Solution contains magnetic nano beads. Please shake well before use. 6. Place the tube in MagListo -2 (mini)/ MagListo -15 (midi, maxi) Magnetic Separation Rack with the magnet plate attached and invert the tube 3~4 times gently until the beads tightly bind to magnet. - Attachment Combine the magnet plate to the stand. 7. Without removing the tube from MagListo rack, carefully pour the supernatant out and completely remove the remaining supernatant using paper towel by blotting. 20

24 - Discard solution Discard solution by inverting the MagListo rack. The silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discard solution, invert the rack completely for the solution not to smear on the rack. 8. (1 st washing: 8-10) Detach the magnet plate from MagListo rack. Add 700 μl (mini)/ 5 ml (midi)/ 10 ml (maxi) of Buffer 4 (2 nd Washing) to the tube. Close the cap and mix by vortex mixer until beads are fully resuspended. - Detachment Push up the magnet plate gently. 9. Attach the magnet plate to MagListo stand and invert the tube 3 ~ 4 times gently until the beads tightly bind to magnet. 10. Without removing the tube from MagListo rack, pour the supernatant out and remove the remaining supernatant using paper towel by blotting. 11. Go to step 15 of DNA Extraction from Animal Tissue in page 16 and continue the clean-up process. 21

25 XIII. Troubleshooting guide Comments and suggestions Buffers or other reagents may have been exposed to external changes or conditions that reduce its effectiveness. Please make sure that reagents were stored at room temperature at all times upon arrival and all reagent bottles were closed tightly, in order to preserve ph and stability, and to avoid contamination. Please check and add ethanol to the Buffer 3 (1 st Washing) and 4 (2 nd Washing). After adding ethanol, mix Washing Buffer well and always mark the Washing Buffer bottles. If not, Washing Buffer remain concentrated and may wash away the adsorbed DNA. Low yield of DNA The lysis may have been incomplete especially in case of tissue. Please, ensure that the sample changes clarity from turbid to clear, indicating that protein digestion has occurred. Extend the incubation time if unlysed tissue is still present. The amount of time for complete lysis varies depending on the type of tissue or sample type used. If a gelatinous mass still remains after the overnight incubation, centrifuge the sample and take the supernatant only for extraction of DNA. A shaking water bath should be used for efficient lysis. There is too much starting material to extract DNA. Appropriate amount of starting material (see product specification in page 4) should be used for efficient extraction of genomic DNA. Beads may have been dried insufficiently. You must always completely dry beads in a drying step. Remained ethanol can decrease the purity of DNA. Low A 260/280 ratio Take enough time to completely dry the beads. Incomplete suspension of beads during the washing step causes salts to remain in the purified DNA. Make the beads suspended thoroughly during the washing process. 22

26 MagListo 5M Genomic DNA Extraction Kit copurify DNA and RNA when both are present in the sample. If RNA-free genomic DNA is required, RNase A Copurification of RNA should be added to the sample before addition of Buffer 2 (Binding). If you want to remove RNA in the eluate, refer to DNA Clean-Up protocol in page 20 for details. Aggregation of Magnetic Nano Bead There is an excess amount of sample to extract DNA. Appropriate amount of starting material (see product specification in page 4) should be used for efficient extraction of genomic DNA. There is a white precipitate in some buffer A white precipitate may form in Buffer l (Lysis) or Buffer 2 (Binding) after prolonged storage at low temperature. Incubating at 60 should dissolve any precipitate in Buffer l (Lysis) or Buffer 2 (Binding). 23

27 XIV. Ordering Information Cat no. Product Description Size K-3601SM MagListo TM 5M Plamid Extraction Kit, 8 reactions (mini) 1 kit K-3601 MagListo TM 5M Plamid Extraction Kit, 100 reactions (mini) 1 kit K-3600 MagListo TM 5M Plamid Extraction Kit, 500 reactions (mini) 1 kit K-3602 MagListo TM 5M Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit K-3603 MagListo TM 5M Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3604 MagListo TM 5M Plant Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit K-3605 MagListo TM 5M Plant Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3606 MagListo TM 5M Gel Extraction Kit, 8 reactions (mini) 1 kit K-3607 MagListo TM 5M Gel Extraction Kit, 100 reactions (mini) 1 kit K-3608 MagListo TM 5M PCR Purification Kit, 8 reactions (mini) 1 kit K-3609 MagListo TM 5M PCR Purification Kit, 100 reactions (mini) 1 kit K-3610 MagListo TM 5M Cell Total RNA Extraction Kit, 8 reactions (mini) 1 kit K-3611 MagListo TM 5M Cell Total RNA Extraction Kit, 100 reactions (mini) 1 kit K-3614 MagListo TM 5M Forensic Sample DNA Extraction Kit, 8 reactions (mini) 1 kit K-3615 MagListo TM 5M Forensic Sample DNA Extraction Kit, 100 reactions (mini) 1 kit TM-1000 MagListo TM -8Ch Magnetic Separation Rack 1 ml tube x 8 holes TM-1010 MagListo TM -2 Magnetic Separation Rack 2 ml tube x 8 holes TM-1020 MagListo TM -15 Magnetic Separation Rack 15 ml tube x 6 holes TM-1030 MagListo TM -50 Magnetic Separation Rack 50 ml tube x 3 holes TM-1040 MagListo TM -96 Magnetic Separation Rack 96-well plate 1ea TM-1100 MagListo TM Magnetic Separation Rack Bundle Set MagListo TM -2,-15,-50, and -96 (4 racks, 1 each) K-3601-A Blow Dryer 1 ea HT-15-NG 1.5 ml microcentrifuge tube 500 ea / pk HT-20-NG 2 ml microcentrifuge tube 500 ea / pk 24

28 XIV. Explanation Symbols Catalog Number Contains sufficient for (n) tests USE BY Consult Instruction For Use Batch code Caution, consult accompanying documents Temperature Limitation In Vitro Diagnostics Medical Device Manufacturer Caution, Potential Biohazard DO NOT REUSE Authorized Representative in the European Community 25

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